Development and Validation of Bioanalytical Method for Determination of Nebivolol and Valsartan in Human Plasma by Using RP-HPLC Cover

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Development and Validation of Bioanalytical Method for Determination of Nebivolol and Valsartan in Human Plasma by Using RP-HPLC

European Pharmaceutical Journal

Volume 68 (2021): Issue 2 (December 2021)

By: R. N. Kachave and A. G. Mundhe

Open Access

|Mar 2022

Figures & Tables

Structure of Nebivolol.

Structure of Valsartan.

Chromatogram of Optimized Chromatographic Method.

Extracted standard blank sample.

Calibration Curve of Nebivolol.

Calibration Curve of Valsartan.

j_afpuc-2021-0019_tab_010

ABBREVIATIONS	FORMS
NL	Nebivolol
VL	Valsartan
RP	Reverse phase
HPLC	High-performance liquid chromatography
BA	Bioavailability
BE	Bioequivalence
API	Active pharmaceutical ingredient
UPLC	Ultra-performance liquid chromatography
UV	Ultraviolet
QC	Quality control
RT	Retention time
USP	United States Pharmacopeia
CV	Coefficient of variation
IS	Internal standard
SD	Standard deviation

Matrix samples’ stability of nebivolol and valsartan at −70°C ± 5°C for 37 days_

Parameters	Nebivolol				Valsartan
Parameters	HQC (comparison sample)	HQC (stability sample)	LQC (comparison sample)	LQC (stability sample)	HQC (comparison sample)	HQC (stability sample)	LQC (comparison sample)	LQC (stability sample)
n	6	6	6	6	6	6	6	6
Mean	7.9880	8.0145	1.4953	1.4902	6386.5000	6401.5000	1194.5000	1188.6667
SD	0.02458	0.05858	0.02273	0.01646	22.29574	21.37054	17.14351	14.17980
% CV	0.31	0.73	1.52	1.10	0.35	0.33	1.44	1.19
% Mean accuracy	99.85	100.18	99.69	99.34	99.79	100.02	99.54	99.06
% Mean stability	100.33		99.65		100.23		99.51

Observation of optimised method chromatogram_

System suitability parameters	Atorvastatin	Nebivolol	Valsartan
RT	2.597	3.189	4.374
Area	97,579	5671	11,302
USP plate count	9792.8	9135.0	8011.3
USP tailing	1.4	1.4	1.1
USP resolution	-	4.6	6.9
Area ratio	-	0.05686	0.3545
SD	-	0.0316	1.00
% CV	-	0.0512	1.18

Matrix factor evaluation of nebivolol and valsartan_

Parameters	Nebivolol		Valsartan
Parameters	HQC	LQC	HQC	LQC
n	18	18	18	18
Mean	7.9592	1.4937	6400.5556	1190.4444
SD	0.07785	0.01768	44.02035	30.91016
% CV	0.98	1.18	0.69	2.60
% Mean accuracy	99.49	99.58	100.01	99.20
No. of QC failed	0	0	0	0

Intra-day precision and accuracy of nebivolol and valsartan_

Parameters	Nebivolol				Valsartan
Parameters	HQC	MQC	LQC	LLOQ QC	HQC	MQC	LQC	LLOQ QC
n	6	6	6	6	6	6	6	6
Mean	7.9513	4.9662	1.4943	0.4985	6403.1667	4017.5000	1196.000	396.5000
SD	0.05211	0.10071	0.01707	0.01320	10.60974	107.61924	11.6666	8.06846
% CV	0.66	2.03	1.14	2.65	0.17	2.68	0.97	2.03
% Mean accuracy	99.39	99.32	99.62	99.70	100.05	100.44	100.44	99.13

Observation table for linearity of nebivolol and valsartan_

Nebivolol			Valsartan
Final conc. in ng/mL	AUC	Area ratio	Final conc. in ng/mL	AUC	Area ratio
0.5	570	0.006	400	3415	0.0350
1	1235	0.013	800	6724	0.0689
1.5	1785	0.018	1200	10,160	0.1041
4	4643	0.048	3200	26,678	0.2731
5	5880	0.060	4000	34,216	0.3506
6	6902	0.071	4800	41,162	0.4217
8	9176	0.094	6400	52,728	0.5402
10	11,175	0.115	8000	67,420	0.6910
Parameters	Nebivolol		Valsartan
Correlation coefficient	0.998		0.9994
Regression equation	y = 1140×		8.4073×
Linearity range	0.5–10 ng/mL		400–8000 ng/mL

Recovery study of nebivolol and valsartan_

Parameters	Nebivolol						Valsartan
Parameters	Unextracted (HQC)	Extracted (HQC)	Unextracted (MQC)	Extracted (MQC)	Unextracted (LQC)	Extracted (LQC)	Unextracted (HQC)	Extracted (HQC)	Unextracted (MQC)	Extracted (MQC)	Unextracted (LQC)	Extracted (LQC)
n	6	6	6	6	6	6	6	6	6	6	6	6
Mean	9445	9253	5965	5782	1886	1857	54,658	53,310	35,376	34,761	10,665	10,507
SD	59.17	70.20	53.57	66.05	12.32	31.02	657.59	400.50	416.36	210.54	143.61	70.53
% CV	0.62	0.75	0.89	1.14	0.65	1.67	1.20	0.75	1.17	0.88	1.34	0.67
% Mean recovery	97.96		96.93		98.46		97.54		98.26		98.52
Overall % mean recovery	97.784						98.112

Inter-day precision and accuracy of nebivolol and valsartan_

Parameters	Nebivolol				Valsartan
Parameters	HQC	MQC	LQC	LLOQ QC	HQC	MQC	LQC	LLOQ QC
n	6	6	6	6	6	6	6	6
Mean	7.9238	4.9362	1.4907	0.4977	6394.3333	3999.0000	1194.8333	393.5000
SD	0.08962	0.08039	0.01770	0.01799	15.83246	70.11419	11.72035	6.05805
% CV	1.13	1.63	1.19	3.61	0.25	1.75	0.98	1.54
% Mean accuracy	99.05	98.72	99.38	99.53	99.91	99.98	99.57	98.38

Long-term stock solution stability of nebivolol and valsartan_

Parameters	Nebivolol		Valsartan
Parameters	HQC	LQC	HQC	LQC
n	6	6	6	6
Mean	7.9938	1.4927	6391.6667	1193.5000
SD	0.01950	0.02082	21.42584	20.08731
% CV	0.24	1.39	0.34	1.68
% Mean accuracy	99.92	99.51	99.87	99.46

Matrix samples’ stability of nebivolol and valsartan at −20°C ± 5°C for 37 days_

Parameters	Nebivolol				Valsartan
Parameters	HQC (comparison sample)	HQC (stability sample)	LQC (comparison sample)	LQC (stability sample)	HQC (comparison sample)	HQC (stability sample)	LQC (comparison sample)	LQC (stability sample)
n	6	6	6	6	6	6	6	6
Mean	7.9930	7.8872	1.4872	1.4898	6394.5000	6391.1667	1193.1667	1188.5000
SD	0.02481	0.15394	0.02729	0.01393	23.57753	18.50856	20.48821	18.98157
% CV	0.31	1.95	1.83	0.94	0.37	0.29	1.72	1.60
% Mean accuracy	99.91	98.59	99.14	99.32	99.91	99.86	99.43	99.04
% Mean stability	98.68		100.18		99.95		99.61

Batch-to-batch precision and accuracy of nebivolol and valsartan_

Parameters	Nebivolol				Valsartan
Parameters	HQC	MQC	LQC	LLOQ QC	HQC	MQC	LQC	LLOQ QC
n	18	18	18	18	18	18	18	18
Mean	7.9577	4.9657	1.4917	0.4981	6398.5000	3993.7222	1194.17	396.1667
SD	0.07478	0.08347	0.01596	0.01470	11.92254	79.93071	11.1596	7.10634
% CV	0.94	1.68	1.07	2.95	0.19	2.00	0.93	1.79
% Mean accuracy	99.47	99.31	99.44	99.61	99.98	99.84	99.44	99.04

DOI: https://doi.org/10.2478/afpuc-2021-0019 | Journal eISSN: 2453-6725

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Language: English

Page range: 49 - 58

Submitted on: Apr 14, 2018

Accepted on: Jun 1, 2018

Published on: Mar 12, 2022

Published by: Comenius University in Bratislava, Faculty of Pharmacy

In partnership with: Paradigm Publishing Services

Publication frequency: 2 issues per year

Keywords:

Method development,

ICH Guidelines (ICH M10),

Related subjects:

Pharmacy, other

© 2022 R. N. Kachave, A. G. Mundhe, published by Comenius University in Bratislava, Faculty of Pharmacy
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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